The present invention relates to an in vitro selection procedure used to select high menthol producing genotypes from in vitro raised somaclonal variants or among vegetatively propagated large population of Mentha arvensis clones.
The industrial mint crops have found wide cultivation in several countries for the commercial production of essential oils due to the economic importance of the monoterpene components. The essential oil of Mentha arvensis Linn. var Piperascens is a well known source of the monoterpene xe2x80x98mentholxe2x80x99 used in the cosmetics, pharmaceutical, food, confectionery and liquor industries. The biosynthesis of menthol in the plant is regulated in a coordinated cascade fashion during plant differentiation. Specific oil glands (trichomes) formed on the surface of the leaf isolate these toxic monoterpenoid products to prevent the cellular damage. Various methods of breeding using intra and inter-specific hybridisations, clonal selections induced mutagenesis are being attempted for genetic improvement of this plant. But the main impediment is gyanodioecy and polyploid nature which results in the male sterile hybrids which hamper further screening of high menthol strains. Similarly, improvement of the genotypes by somaclonal variations requires the screening of a large number of clones for menthol content. Monoterpenes are cytotoxic to plant tissues and act through inhibition of respiration and photosynthesis by drastically affecting the mitochondria, golgi bodies etc. and decreasing cell membrane permeability. In the present invention for the exploratory experimentation the applicants compared the menthol content in the oil of selected cultivars of Mentha arvensis Linn. var Piperascens released by CIMAP.
These varieties were also checked for the in vitro menthol tolerance level by the regenerated shoots from these genotypes by inoculating on menthol containing medium. The main objective here was to explore the possible relationship between these two characters. Keeping in view the rationale of feed back toxicity by metabolic end product (menthol), the correlation could be utilized as a probing and selective tool to identify high menthol clones.
The applicants have earlier reported a high efficiency protocol for rapid generation, detection and selection of somaclonal variants through molecular approach in Mentha arvensis (S P S Khanuja, A K Shasany, S Dhawan, S Kumar, 1998, Rapid procedure for isolating somaclones of altered genotypes in Mentha arvensis. Journal of Medicinal and Aromamatic Plants Science, 20: 359-361). The applicants have also successfully defined the conditions and media for stable micropropagation of selected clones (A K Shasany, S P S Khanuja, S Dhawan, U Yadav, S Sharma, S Kumar, 1998, High regenerative nature of Mentha arvensis internodes. Journal of Biosciences 23: 641-646).
In the present invention, the applicants utilized the former protocol (Khanuja el at., 1998) to generate and capture the somaclonal variants at molecular level in larger frequencies. The applicants then screened the somaclones for their levels of in vitro tolerance to menthol in poison agar medium by devising a novel method for rapid and dependable selection of tolerant clone(s) right at the tissue culture stage. Further the tolerant plants, which survived were multiplied by the latter protocol (Shasany et al, 1998) and tested for their stability, essential oil, menthol content and biomass yield upto field level
The main object of the invention is to develop an in vitro method for selection of high menthol producing genotypes from somaclonal variants or vegetatively propagated large population of Mentha arvensis clones.
Another object is to provide a method or a table for the identification or selection of menthol-rich genotypes from a large population of Mentha arvensis clones.
Accordingly, the invention provides a method for the selection of menthol rich or high menthol producing genotypes from a large population of Mentha arvensis clones.
Accordingly, tee invention provides a new, simple and rapid in vitro method for selection of menthol rich mint genotypes from a large population of independent clones, comprising the steps of:
(i) raising a heterogeneous population of Mentha arvensis clones in vitro or by vegetative methods,
(ii) transferring the plantlets/shoots to a basal medium containing selective compounds in an amount sufficient to cause toxic effect in more than 95% of time clones,
(iii) selecting surviving clones, their hardening in the glasshouse, transfer to field, reconfirmation of menthol tolerance through repeated in vitro assays, and
(iv) multiplying selected clones and confirming genetic uniformity through RAPD analysis before menthol yield field trial.
In an embodiment, the vegetative methods for raising a heterogeneous population of Mentha arvensis clones in vitro comprise development of suckers/runners, open pollinated seed progeny, plants raised from seeds produced by planned hybridization between selected parents through controlled pollination.
In still another embodiment, the basal medium comprises MS medium containing cytotoxic compounds in a concentration sufficient to cause irreversible wilting, browning, or death of more than 95% of the plants.
In a further embodiment, the compounds for selection are selected from menthol, monoterpenes and such similar compounds. In one embodiment, the most preferred cytotoxic compounds are menthol and monoterpenes.
In another embodiment, the menthol rich mint genotypes yield at least 74% or more menthol in their essential oils.
In yet another embodiment, the screening result can also be used as the rapid indicator of menthol yield different varieties for evaluation purpose.
In yet another embodiment, the selection pressure is the concentration of menthol (50 and 80 xcexcg mlxe2x88x921), which causes the lethality of at least 95% of clones/plantlets and the selection pressure is not limited to menthol as other monoterpenes and cytotoxic compounds can also be used.
In the other embodiment, the compounds for selection are toxic to cytoplasm causing complete loss of chlorophyll in the lower leaves and gradually covering the whole shoot within a week time leading to death of the shoot.
In another embodiment, the compounds for selection are selected from the group comprising nerol, geraniol, citronellol linalool, myrcene, terpineol limolene, cineole, terpinol, terpinene, cymene, thymol, carvone, pinene, fenchol, fenchone, camphene, borneol, camphor, thujone, thujanol, sabinene, carene and other related compounds.
In the other embodiment, the procedure can be carried out in any suitable agar medium with selective compound in sterilizable flasks, tubes or containers of various sizes permitting the proper growth conditions to the shoots/plantlets.
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